Microbubble tether for diagnostic and therapeutic applications

ABSTRACT

The present disclosure relates to a composition of albumin microbubbles to which are bound one or more moieties that exhibit a binding preference for the albumin microbubbles relative to free, native HSA. Production of the albumin microbubble composition and use of the albumin microbubble composition in ultrasound mediated delivery of therapeutic or diagnostic agents is also discussed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 14/550,689, filed on Nov. 21, 2014, entitled Microbubble Tetherfor Diagnostic and Therapeutic Applications.

BACKGROUND

The subject matter disclosed herein relates to the use of microbubblecompositions in diagnostic or therapeutic applications.

A variety of therapeutic agents or drugs are not commercially ormedically viable due to one or more of adverse side effects, poorsolubility in blood, or high cost. To address certain of these failings,delivery systems such as ultrasound microbubble (USMB) mediated deliverysystems have been developed. In such a system, a gas-filled microbubbleis associated with a treatment agent of interest. Under treatmentconditions, the gas-filled microbubbles undergo cavitation in responseto the application of ultrasonic energy at a targeted site (i.e.,anatomic region) of interest. This cavitation event results inmicrobubble destruction (inertial cavitation) and is, presumably,accompanied by a shock wave that leads to the formation of transientpores in the membranes of surrounding tissues and cells. The transientpores allow the treatment agent to gain access to the tissues to betreated. In other scenarios, ultrasound results in stable cavitationrather than inertial cavitation, which can also lead to enhanceddelivery of drugs to tissues or cells. Such approaches have beeninvestigated with possible future applications involving the targeteddelivery of small molecule drugs, oligonucleotides, and plasmid DNA(pDNA), such as to a patient.

Delivery of therapeutic agents can be significantly enhanced in thepresence of microbubbles upon application of ultrasound. Generally,agent pharmacokinetic properties and clearance mechanisms are keydrivers behind delivery to tissue and cells. Binding between themicrobubble and the agent of interest can affect the efficiency of agentdelivery to a site of interest by, for example, enhancing in vivostability, manipulating agent biodistribution characteristics, or othermechanisms. The nature and extent of such binding relationships, andtheir effect on delivery, however, have generally not been thoroughlyresearched and are poorly understood. It may be desirable, therefore, todevelop a more suitable USMB mediated drug delivery system andassociated delivery agents.

BRIEF DESCRIPTION

In one embodiment, a composition is provided. The composition includes amicrobubble comprising an albumin shell, and a moiety bound to themicrobubble. The moiety binds preferentially to the microbubble relativeto native albumin.

In an additional embodiment, a composition is provided. The compositionincludes an albumin microbubble comprising an albumin shell. Thecomposition also includes a therapeutic or diagnostic agent, and acyanine 5 derivative linking the albumin microbubble and the therapeuticor diagnostic agent.

In a further embodiment, a method is provided. The method includes themixing at room temperature of an albumin microbubble composition and thedrug agent or moiety-bound drug agent wherein the drug agent or themoiety-bound drug agent binds preferentially to the microbubble relativeto native albumin.

In yet a further embodiment, an ultrasound-based treatment method isprovided. The method includes the act of introducing an albuminmicrobubble composition into a patient. The albumin microbubblecomposition comprises: an albumin shell, a molecule of interest, and amoiety binding the molecule of interest to the albumin shell. The moietypreferentially binds to the albumin shell relative to free, native humanserum albumin. A further act of the method is then to direct ultrasonicenergy at an anatomic region of interest to cause cavitation of thealbumin microbubbles at the anatomic region of interest.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood when the following detaileddescription is read with reference to the accompanying drawings in whichlike characters represent like parts throughout the drawings, wherein:

FIG. 1 depicts an ultrasound system suitable for use in an ultrasoundmicrobubble mediated drug delivery system, in accordance with aspects ofthe present disclosure;

FIG. 2 depicts a sample preparation process flow, in accordance with oneaspect of the present disclosure;

FIG. 3 depicts chemical structures of molecules under study, inaccordance with aspects of the present disclosure;

FIG. 4 depicts chemical reactions used to produce certain of themolecules of FIG. 3;

FIG. 5 depicts a further sample preparation process flow, in accordancewith one aspect of the present disclosure;

FIG. 6 depicts gray scale representations of brightfield andfluorescence microscopy results for a first composition, in accordancewith aspects of the present disclosure;

FIG. 7 depicts gray scale representations of brightfield andfluorescence microscopy results for a second composition, in accordancewith aspects of the present disclosure;

FIG. 8 depicts gray scale representations of brightfield andfluorescence microscopy results for a third composition, in accordancewith aspects of the present disclosure;

FIG. 9 depicts gray scale representations of brightfield andfluorescence microscopy results for a fourth composition, in accordancewith aspects of the present disclosure;

FIG. 10 depicts gray scale representations of brightfield andfluorescence microscopy results for a fifth composition, in accordancewith aspects of the present disclosure;

FIG. 11 depicts a graphical representation of fluorescence microscopyresults, in accordance with aspects of the present disclosure;

FIG. 12 depicts a graphical representation of flow cytometry results, inaccordance with aspects of the present disclosure;

FIG. 13 depicts a further sample preparation process flow, in accordancewith aspects of the present disclosure;

FIG. 14 depicts graphical representations of dye binding fractionprofiles as a function of native HSA concentration and as a function ofmicrobubble albumin concentration, in accordance with aspects of thepresent disclosure; and

FIG. 15 illustrates another sample preparation process flow which may beused to prepare therapeutic compositions in accordance with aspects ofthe present disclosure.

DETAILED DESCRIPTION

Certain aspects of the present disclosure relate to a diagnostic ortherapeutic microbubble composition that includes a moiety thatfunctions as a binding tether to albumin microbubbles. The moiety may beattached to a pharmacological or diagnostic agent, such as a therapeuticagent or a diagnostic composition that may facilitate imaging or otherdiagnostic procedures at a target location. Further, in certainembodiments, the moiety may also function as an optical tracer (e.g.,may fluoresce) and, in such embodiments, the optical tracing may beleveraged for diagnostic purposes, e.g., as an optical probe. A mediatedagent delivery system suitable for in vivo use with such compositionsmay, therefore, include: 1) albumin microbubbles, 2) one or moremoieties that show a binding preference for the albumin microbubbles(e.g., Optison™) relative to free native human serum albumin, and 3)ultrasound energy for targeted delivery of the therapeutic or diagnosticcomposition. In some embodiments, the one or more moieties may bemolecules used to functionalize a therapeutic or diagnostic compositionof interest. In other embodiments, the moiety may be an intrinsic groupof the therapeutic or diagnostic composition of interest such that thecomposition itself exhibits the binding preference for the albuminmicrobubbles relative to free native human serum albumin.

With respect to the albumin microbubbles, these microbubbles may bederived from human serum albumin (HSA) or from other suitablebiocompatible albumin sources, including synthetic or synthesizedalbumin. An example of one such albumin microbubble source is Optison™albumin shell microbubbles. Optison™, or other suitable microbubbleforming media, may be derived from HSA but may be distinct from free,native HSA found in vivo in the body. By way of example, in the contextof Optison™ microbubbles, these microbubbles have an HSA shellsurrounding a perfluoropropane core. In some embodiments, a suitablemicrobubble may have a mean diameter in the range of about 3-4.5 μm(e.g., between about 1 μm and about 10 μm). The albumin present ingas-filled, albumin microbubble compositions such as Optison™ or othersuitable compositions may be distinct from HSA found in vivo for one ormore of the following reasons: it may be partially denatured, it may bepartially crosslinked or the albumin may be present in a different ratioof one or more protein conformations relative to native HSA.

As noted above, in certain embodiments the one or more moieties thatattach to the microbubble may exhibit a binding preference for albuminmicrobubbles relative to free native human serum albumin. As discussedherein, in certain embodiments molecules that show a binding preferenceto albumin microbubbles relative to free, native HSA can be provided asfunctional groups on drugs, binding them to the microbubbles andenabling efficient drug delivery in vivo upon application of ultrasoundenergy. In certain instances, the moiety may intrinsically be present onthe drug of interest, and thus need not be added in a separate step. Inone embodiment, the moiety component may be a pegylated or non-pegylatedcyanine 5 (Cy5) derivative that can be used to “tether” a therapeutic ordiagnostic agent to the microbubbles. For example, a Cy5 derivativeacting as a tether to a microbubble may be functionalized with atherapeutic agent, such as a small molecule drug, an oligonucleotide(such as siRNA), or a large molecule (such as a plasmid DNA).

With the preceding in mind, present approaches may be used to addressdrug delivery issues related to adverse side effects, poor solubility inblood, or high cost. In one implementation, by binding a therapeuticagent to a microbubble (such as using a tether), a complex is formedthat reduces the drug's bioavailability as it circulates in vivo, untilit is released at the target site upon sonication. This has the effectof reducing adverse side effects. The complex formation can also enhancesolubility, as a microbubble can carry multiple copies of a drug thatmight otherwise exhibit poor solubility. Overall cost is reduced becausethe efficiency of drug delivery can in principle be significantlyenhanced upon binding to bubbles by increasing local drug concentrationat the site of ultrasound delivery.

Further, and as noted above, in certain embodiments, the moiety (e.g.,Cy5 derivatives) fluorescently tags the microbubble to which they areattached. In such embodiments, the Cy5 derivative may alternatively oradditionally function as an optical tracer, allowing localization orvisualization of the microbubbles through non-invasive imaging.

With the preceding in mind, and turning to FIG. 1, an example ofultrasound system components that may be present in an USMB mediateddrug delivery system 100 is depicted. As will be appreciated, suchcomponents may correspond to those found in a conventional ultrasoundimaging system and the system may in practice be used to image thetarget tissue in conjunction with the mediated delivery of a therapeuticagent. In the depicted example, the ultrasound system includes anultrasound probe 102 and an ultrasound console 104 suitable forgenerating and receiving ultrasound signals via the probe 102. Incertain embodiments, the ultrasound console 104 may include beam-formersand image reconstruction and processing circuitry used to direct theultrasonic energy 106 into the tissue 108 of the patient and toreconstruct the return signals measured at the probe 102. For example,the ultrasound console 104 may control the strength, beam focus orforming, duration, phase, and frequency of the ultrasound signalstransmitted by the ultrasound probe 102, and may decode the informationcontained in the plurality of reflected ultrasound signals from thetissue to a plurality of discernible electrical and electronic signals.In USMB embodiments, the ultrasonic energy 106 may, within the targetedtissue 108, facilitate the release of a therapeutic or diagnostic agentat an anatomic location of interest. The return signals may be processedat the console 104 to generate images of the anatomic region of interestunderlying the probe 102.

The ultrasound system 100 may also include an operator interface 110allowing a user to interact with and control the console 104. Theoperator interface 110 may include a keyboard, a mouse, and other userinteraction devices. The operator interface 110 can be used to customizea plurality of settings for an USMB procedure, to effect system levelconfiguration changes, and to allow operator activation and operation ofthe ultrasound system 100.

In the depicted example, the operator interface 110 is connected to theultrasound console 104, a display module 112, and a printer module 114,some or all of which may be provided as an ultrasound workstation. Thedisplay module 112 receives image information from the ultrasoundconsole 104 and presents the image of the objects underlying theultrasound probe 102. The printer module 114 is used to produce a hardcopy of the ultrasound image in either gray-scale or color. In general,the reflected ultrasound signals and corresponding images conveyinformation about thickness, size, and location of various tissues,organs, tumors, and anatomical structures in relation to transmittedultrasound signals.

With the preceding in mind, certain studies related to the presentsubject matter are discussed below. With respect to the existence ofmoieties having a binding preference for albumin microbubbles relativeto free, native HSA, a study was performed to characterize the bindingaffinity between the ApoA-I pDNA and albumin microbubbles (e.g.,Optison™ bubbles). FIG. 2 illustrates the sample preparation method usedin this study. In this example, three distinct operations are shown. Inthe first operation (steps 200), bubble separation and a wash to removefree albumin were performed. In the second operation (step 218), thewashed bubbles were mixed with the pDNA/dye binding solution. In thethird operation (steps 202), two saline or two native human serumalbumin washes were performed followed by saline reconstitution toafford the final composition for analysis.

With respect to the first operation, in this example, Optison™ was usedas the microbubble composition, and it was washed once with saline (step212) to remove free HSA present in the commercial product. Inparticular, in the depicted example, the microbubble product 206 wasinitially centrifuged (step 208), the bottom layer removed (step 210),saline added and mixed (step 212), followed by an additionalcentrifugation (step 214). After centrifugation the bottom layer wasagain removed, and to the remaining albumin microbubbles was mixed theDNA/dye composition (step 218).

With respect to the DNA/dye composition, in this study, pDNA was firstmixed with propidium iodide (PI), which fluoresces when intercalatedinto the DNA structure but does not fluoresce as the result of anyinteraction with the microbubble (should one exist). As noted above, thepDNA/dye solution was mixed with the albumin microbubbles (step 218) andthe product centrifuged (step 220). The resulting microbubble/DNA/dyeproduct was washed twice (steps 224 and 226) with a 9.5 mg/mL native HSAsolution followed by a saline reconstitution (step 228) to yield thefinal analytic product 230. In other experimental runs, differentwashing sequences were employed.

In this study, the affinity of the pDNA for the microbubble as afunction of bubble washing was assessed using fluorescence microscopyand flow cytometry. No evidence for statistically relevant bindingbetween the pDNA and the albumin microbubble was observed. Inparticular, any binding interaction between the pDNA and themicrobubbles was sufficiently weak that a simple microbubble separationfollowed by a saline wash was sufficient to remove the pDNA.

In contrast, other studies were performed in which the agent in questionwas found to bind to the microbubble. For example, in one set of studiesthe model agents investigated were derivatives of cyanine 5 (Cy5), suchas pegylated Cy5 dyes. Generally, pegylated and non-pegylated agentscomprising hydrophobic Cy5 moieties were observed to be good binders toalbumin microbubbles, whereas agents comprising highly sulfonated Cy5groups (Cy5**) were observed to be poor binders to albumin microbubbles.

Turning to FIG. 3, examples of molecules which were investigated areshown. The molecules investigated included the Cy5 and Cy5** free acids(referred to as Cy5COOH and Cy5**COOH) as well as derivativessubstituted with polyethyleneglycol (PEG) (to modify the size of thetarget) having average molecular weights of 550 g/mole and 20,000g/mole. The Cy5COOH was commercially available and the Cy5**COOH wassynthesized by GE Healthcare. The pegylated molecules were synthesizedfrom the respective free acids as shown in equations 1 and 2 of FIG. 4.The absorption maxima were determined to be different for the twoclasses of Cy5 dyes. Studies indicated that the optimal excitationwavelength to achieve maximum fluorescence intensity for theunsulfonated Cy5 is 640 nm (with emission at 655 nm) and for Cy5** it is650 nm (with emission at 670 nm). The quantum yield of unsulfonated Cy5has been determined to be 1.6 times that of Cy5**.

In the studies, dye/bubble mixtures using Cy5 were prepared as shown inFIG. 5. In the depicted process, three operations are shown. In thefirst operation (steps 200), bubble separation and a wash to remove freealbumin were performed. In the second operation (step 240), the washedbubbles were mixed with the dye binding solution. In the third operation(steps 242), a sequence of wash steps were performed followed by salinereconstitution (step 250) to afford the final composition for analysis.

With respect to the first operation, in this example, Optison™ was usedas the microbubble composition 206, and it was washed once with saline(step 212) to remove free HSA present in the commercial product. Inparticular, in the depicted example, the microbubble product 206 wasinitially centrifuged (step 208), the bottom layer removed (step 210),saline added and mixed (step 212), followed by an additionalcentrifugation (step 214). After centrifugation the bottom layer wasagain removed, and to the remaining albumin microbubbles was mixed theCy5 dye composition (step 240). In the tested scenarios, the Cy5derivative of interest and the albumin microbubble formulation weremixed at room temperature. The product derived from mixing the dyesolution and microbubbles was centrifuged (step 220).

The resulting microbubble/dye product was washed (steps 244, 246, 248)with one or more wash solutions. A variety of wash scenarios weretested. In the first scenario, three saline washes were performed. Inthe second scenario, two denatured HSA washings and one saline wash wereperformed. In the third scenario, two native HSA washes and one salinewash were performed. Protein washes contained 9.5 mg/mL HSA. Thedenatured HSA used in these experiments was soluble, homogeneous HSAisolated from commercial Optison™ product. The washed product wasreconstituted in saline (step 250) to yield the final analytic product252.

The resulting analytic products 252 were used to assess the bindingaffinity between the Cy5 derivatives and the albumin microbubbles. Inparticular, in certain studies two complementary fluorescence-basedapproaches were used to characterize binding in the finaldye/microbubble mixtures: fluorescence microscopy and flow cytometry.

In the fluorescence microscopy approach, images from multiple fields ofview were acquired and segmented, the background intensity wassubtracted, and the number, size, and brightness of the remainingfeatures were calculated. These measured values were believed tocorrespond to the Cy5 dye bound to the imaged microbubbles.

In the flow cytometry approach, microbubbles pass through a laserintercept one at a time as a consequence of hydrodynamic focusing. Themicrobubbles scatter the laser light, which is registered as an event.Any fluorescent molecules (e.g., the Cy5 dye derivatives) associatedwith a given microbubble absorb the laser light and the resultingfluorescence is measured. Fluorescence is only measured when there is acorresponding scattering event. In the present studies, the excitationwavelength employed was 633 nm, and differences in quantum yieldsbetween the Cy5 molecules and the Cy5** compounds was taken intoaccount.

FIGS. 6-10 depict the resulting brightfield and fluorescence images ofthe albumin microbubble and Cy5 dye mixtures. The fluorescence images(bottom row images) were captured after subtraction of the backgroundsignal, as noted above. FIG. 6 depicts images acquired using the 20 kPEG Cy5 moiety; FIG. 7 depicts images acquired using the 550 PEG Cy5moiety; FIG. 8 depicts images acquired using the Cy5 COOH moiety; FIG. 9depicts images acquired using the 20 k PEG Cy5** moiety; and FIG. 10depicts images acquired using the 550 PEG Cy5** moiety.

As shown, each fluorescence image was captured in the same field of viewas the corresponding brightfield image above it. Using images capturedin multiple fields of view, the average fluorescence intensity of eachbubble after subtracting the background signal was calculated for eachmixing/washing scenario. A graphical summary of calculated values isshown in FIG. 11. In the graphical summary, the respective error barscorrespond to ±1 standard deviation. As can be seen in the reproducedimages, the Cy5 derivatives exhibited persistent binding. Conversely,the weaker binding Cy5** compounds were easily washed away from thebubbles.

With respect to the flow cytometry results, flow cytometry was used tocharacterize binding in mixtures of albumin microbubbles and Cy5 dyederivatives both before and after the various washing routines, asdescribed above. The results of the flow cytometry study are graphicallysummarized in FIG. 12, where characterization of the microbubble/dyemixtures is shown as a function of wash cycles and where “nHSA”corresponds to native human serum albumin and “dHSA” corresponds todenatured human serum albumin.

Consistent with the fluorescence microscopy studies, the flow cytometryresults indicate that derivatives comprising the hydrophobic Cy5 showbinding to the microbubbles which persists through saline and proteinwashes. Conversely, the hydrophilic Cy5** derivatives are present in amuch lower concentration in washed samples. In particular, in the flowcytometry results, under all wash conditions, the smallest mosthydrophobic dye, the Cy5COOH, showed the highest level of bubblebinding. Consistent with the fluorescence microscopy results, the 20kPEGCy5 showed greater binding than the 550PEGCy5 following salinewashes, but they were essentially equivalent following protein washes.

As will be appreciated, in all of the fluorescence microscopy and flowcytometry studies described, dye/microbubble washings were performedwith either saline solutions or solutions of HSA. Generally, it wasobserved that the Cy5 derivatives bound persistently to the bubblethrough wash cycles employing not only saline, but also native anddenatured HSA. Further, persistent binding of Cy5 derivatives wasobserved when washes were performed using human blood serum or whole(rat) blood. In particular, it was observed overall that unsulfonatedCy5 bound measurably and durably to albumin microbubbles.

In the examples described herein, the albumin binding moiety was mixedwith the albumin microbubbles after microbubble manufacture. As will beappreciated, however, the albumin binding moiety may be present at orincluded in the microbubble manufacturing process so as to be present inor bound to the microbubble product. That is, these steps need not beseparate and discrete, though they are discussed as such in the presentdocument so as to simplify explanation. Thus, implementations in whichthe albumin binding moiety is incorporated into the bubble manufacturingprocess are also considered within the scope of this disclosure.

To gain additional insight into the extent to which the Cy5 and Cy5**derivatives bind to albumin microbubbles, a series of equilibriumstudies were performed. Aspects of these studies helped to: (1)characterize system behavior under conditions more relevant to the invivo experiment, i.e. in the presence of a large excess of native HSA,and (2) characterize the relative affinity that fluorescent agents ofinterest have for HSA in microbubbles compared to native HSA. In theseexperiments, different albumin concentrations (native or present inmicrobubbles) were treated with a fixed concentration of dye (i.e., Cy5derivative). The albumin/dye mixtures were centrifuge filtered toseparate bound dye from unbound, and the fluorescence of the unbound dyewas measured using a plate reader.

The sample preparation method for the experiments using bubbles issummarized in FIG. 13. With respect to the sample preparation steps,bubble separation and a wash to remove free albumin (steps 200) wereperformed. In this example, the microbubble product 206 was initiallycentrifuged (step 208), the bottom layer removed (step 210), salineadded and mixed (step 212), followed by an additional centrifugation(step 214). After centrifugation the bottom layer was again removed(step 280) to yield a microbubble product 282. To perform theequilibrium calculations, a quantification step 284 was performed on themicrobubble product 282 prior to adding (step 290) the respective Cy5derivative under investigation. The resulting microbubble/dyecomposition 292 was then centrifuged, filtered, and analyzed on a platereader (step 294).

With respect to the binding equilibrium between the dye and themicrobubbles, the equilibrium equation:X=n*[HSA]/(K _(d)+[HSA])  (1)was employed, where X is the fraction of ligand bound (based on observedfluorescence), and n is the number of binding sites per HSA. In theequilibrium expression, n (e.g., the number of Cy5 or Cy5** bindingsites) is assumed to be 1.

With the preceding in mind, the fraction of the 20 k PEG Cy5 dye boundto native HSA was measured over a range of albumin concentrations andcompared to the binding fraction measured in mixtures of the dye withalbumin microbubbles. The results are shown graphically in FIG. 14 whichdepicts the dye (e.g. Cy5) binding fraction profiles as a function ofnative HSA concentration (on the left) and as a function of microbubblealbumin concentration (on the right). The corresponding calculatedequilibrium constants are shown in Table 1 below, where the ratio is theratio of binding constant to microbubble albumin versus free native HSAand where HSA (microbubble) refers to the albumin that makes up thealbumin microbubble. Kd denotes the dissociation constant, and Kadenotes the association constant.

TABLE 1 HSA Agent HSA (microbubble) Ratio 20k PEG Cy5 Kd (μM) 1.4 ± 0.20.3 ± 0.1 ~4.5 Ka (μM) 0.7  3.3 20k PEG Kd (μM) 2.2 ± 0.4 0.5 ± 0.0 ~4.5Cy5** Ka (μM) 0.46 2  

As can be seen both in FIG. 14 and Table 1, the binding constant for[dye/albumin in the microbubble shell] is more than 4.0 times higherthan it is for [dye/native HSA] for both the 20 kPEGCy5 and the weakerbinder 20 kPEGCy5**. This is a surprising result in that the bindingsubstrate in both instances is albumin, with the only distinction beingthat albumin in the form of a microbubble shell is preferentially boundrelative to free, native HSA. This will likely affect the equilibriumdistribution or partitioning of dye between the bubbles and free albuminin the blood stream. As will be appreciated, other degrees of bindingpreference (e.g., greater than 2.0 or greater than 3.0) may also be ofsignificance and useful for preferentially binding a therapeutic ordiagnostic agent to albumin microbubbles. As the data presented shows,while both the 20 kPEGCy5 and the 20 kPEGCy5** showed a bindingpreference for albumin microbubbles vs free native albumin, they did notshow the same persistence in binding to microbubbles, for example, afterwashing (FIGS. 6-10). In some embodiments, the stronger bindingunsulfonated Cy5 derivatives may be preferred. In other embodiments, theweaker binding highly sulfonated Cy5** derivatives may be preferred.

Calculations based on these results suggest that, under acceptable invivo test conditions (e.g., 1 mL of 2.5 μM dye mixed with 1 mL ofalbumin microbubble product), each microbubble contains on average about420,000 bound dye molecules when the dye is 20 kPEGCy5. On average,about 5.5% of all bubble albumin molecules have a 20 kPEGCy5 bound tothem. In contrast, under the same conditions, when the dye is 20kPEGCy5**, there are on average about 160,000 dye molecules bound toeach bubble, with about 2.2% of all bubble albumin associated with thedye.

In view of these results, therapeutic and/or diagnostic agents ofdifferent sizes may be attached to albumin microbubbles using a moiety(e.g., a “tether” molecule) that exhibits preferential binding to thealbumin microbubble (e.g., non-native HSA or partially denatured andpartially crosslinked HSA) relative to free, native HSA. Without beinglimited by theory, the long range structure of albumin molecules in thebubble shell, driven by intramolecular and intermolecular arrangement aswell as bubble surface shape, may also play a role in observed bindingpreferences. In certain embodiments, the moiety (e.g., Cy5 or a Cy5derivative) may be provided as a functional group on the therapeutic ordiagnostic agent of interest, either as an added group or a groupintrinsically present on the agent of interest. In other embodiments,the therapeutic or diagnostic agent comprises as part of its chemicalstructure a moiety which is not Cy5 or a Cy5 derivative but which has abinding preference for albumin in microbubbles vs free native albumin.Any of these combinations of albumin microbubble, binding moiety(intrinsic or added), and agent may be used in an ultrasound microbubble(USMB) mediated agent delivery system.

One embodiment for preparing such therapeutic compositions is shown inFIG. 15. In the depicted example, an optional wash step 200 may beperformed to remove free albumin before the agent/binder is added (step260). The product is subsequently, optionally washed (steps 262) withanother composition, such as saline solution, to substantially removeunbound agent/binder before administration to the subject (e.g., washstep 264 and separation step 266). In other embodiments, additionalwashes are conducted. In yet other embodiments, no washes are conducted.In embodiments where washes are conducted after the addition of theagent/binder, the composition may then be reconstituted (step 268), suchas using saline, to generate the therapeutic product 270 foradministration to a patient.

As discussed herein, in certain embodiments the moiety exhibiting abinding preference may act as an optical tracer (e.g., a fluorescenttag). In such embodiments, the optical or fluorescent properties of themoiety may be used in diagnostic applications, such as to monitor orvisualize the location of the bound microbubbles relative to an anatomicregion of interest, such as during a USMB procedure. Examples ofmoieties that exhibit preferential binding to albumin microbubbles(relative to free, native HSA) include, but are not limited to Cy5derivatives.

Technical effects of the invention include a composition of albuminmicrobubbles to which are bound one or more moieties that exhibit abinding preference for the albumin microbubbles relative to free, nativeHSA. The one or more moieties may be functionalized with a therapeuticor diagnostic agent. Other technical effects include performing anultrasound microbubble mediated agent delivery using the albuminmicrobubbles bound with the moiety.

This written description uses examples to disclose the invention,including the best mode, and also to enable any person skilled in theart to practice the invention, including making and using any devices orsystems and performing any incorporated methods. The patentable scope ofthe invention is defined by the claims, and may include other examplesthat occur to those skilled in the art. Such other examples are intendedto be within the scope of the claims if they have structural elementsthat do not differ from the literal language of the claims, or if theyinclude equivalent structural elements with insubstantial differencesfrom the literal languages of the claims.

The invention claimed is:
 1. A composition, comprising: an albuminmicrobubble comprising an albumin shell; a therapeutic or diagnosticagent; and a cyanine 5 acid or cyanine 5-PEG linking the albuminmicrobubble and the therapeutic or diagnostic agent.
 2. The compositionof claim 1, wherein the albumin shell comprises partially denatured,partially cross-linked human serum albumin.
 3. The composition of claim1, wherein the therapeutic or diagnostic agent comprises a smallmolecule drug or a nucleic acid structure.
 4. The composition of claim1, wherein the albumin microbubble has a diameter between about 1 μm toabout 10 μm.
 5. An ultrasound-based treatment method, comprising:introducing the albumin microbubble composition of claim 1 into apatient; and directing ultrasonic energy at an anatomic region ofinterest to cause cavitation of the albumin microbubbles at the anatomicregion of interest.
 6. The ultrasound-based treatment method of claim 5,further comprising: visualizing the presence of albumin microbubbles atthe anatomic region of interest based on fluorescence of the moiety. 7.The ultrasound-based treatment method of claim 5, wherein therapeutic ordiagnostic agent comprises a small molecule drug or a nucleic acidstructure.
 8. The ultrasound-based treatment method of claim 5, whereinthe cyanine 5 acid or cyanine 5-PEG preferentially binds to the albuminshell relative to free, native human serum albumin at a ratio greaterthan
 2. 9. The ultrasound-based treatment method of claim 5, wherein thecyanine 5 acid or cyanine 5-PEG preferentially binds to the albuminshell relative to free, native human serum albumin at a ratio greaterthan 4.0.
 10. The composition of claim 1, wherein the compositioncomprises cyanine 5 acid, and the cyanine 5 acid is a sulfonated acid.11. The composition of claim 1, wherein the composition comprisescyanine 5 acid, and the cyanine 5 acid is an un-sulfonated acid.
 12. Thecomposition of claim 1, wherein the composition comprises cyanine 5-PEG,and the cyanine 5-PEG is un-sulfonated.
 13. The composition of claim 1,wherein the composition comprises cyanine 5-PEG, and the cyanine 5-PEGis sulfonated.